US5644045A - X-linked adrenoleukodystrophy gene and corresponding protein - Google Patents

X-linked adrenoleukodystrophy gene and corresponding protein Download PDF

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US5644045A
US5644045A US08/136,277 US13627793A US5644045A US 5644045 A US5644045 A US 5644045A US 13627793 A US13627793 A US 13627793A US 5644045 A US5644045 A US 5644045A
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Jean-Louis Mandel
Patrick Aubourg
Jean Mosser
Claude Sarde
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Institut National de la Sante et de la Recherche Medicale INSERM
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2799/00—Uses of viruses
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    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus

Definitions

  • the present application relates to the identification and isolation of a gene which is responsible for the adrenoleukodystrophy. It further concerns the protein encoded by this gene and their use in diagnostic or therapeutic procedures.
  • Adrenoleukodystrophy is an X-linked disease affecting 1/20,000 males either as cerebral ALD in childhood or as adrenomyeloueuropathy (AMN) in adults. Childhood ALD is the more severe form, with onset of neurological symptoms between 5-12 years of age. Central nervous system demyelination progresses rapidly and death occurs within a few years. AMN is a milder form of the disease with onset at 15-30 years of age and a more progressive course. Adrenal insufficiency (Addison's disease) may remain the only clinical manifestation of ALD.
  • the principal biochemical abnormality of ALD is the accumulation of very long chain fatty acids (VLCFA) because of impaired ⁇ -oxidation in peroxisomes. The normal oxidation of VLCFA-CoA in patients fibroblasts suggested that the gene coding for the VLCFA-CoA synthetase could be a candidate gene for ALD.
  • VLCFA very long chain fatty acids
  • Adrenoleukodystrophy and adrenomyeloneuropathy are characterized by the presence of an abnormal ALD gene, resulting from deletions or other types of mutations including point mutations. The mututions in the gene may nevertheless remain clinically silent or may lead to various phenotypic clinical expression.
  • ALD gene encompasses the gene involved in ALD and also in its adult variant AMN.
  • sequences responsible for ALD or AMN it must be understood that the abnormal form of the ALD gene is involved in the ALD pathology; of course the normal gene (devoid of mutations, especially of deletions) does not cause the disease.
  • isolation refers to the fact that the nucleotide sequence is separated from the other nucleotide sequences of the Xq28 region of the X chromosome when it is purified for instance from a natural source or organism.
  • This isolated nucleotide sequence is also obtainable from synthetic or semi-synthetic sources, according to well-known methods.
  • This sequence can be any type of nucleotide sequence and especially can be selected among DNA, RNA or cDNA.
  • a particular sequence which is referred to according to the invention consists essentially of the human gene responsible for the ALD. This gene accordingly contains both exons and introns and therefore contains both coding sequences of the gene and regulation sequences.
  • the gene coding for the ALD protein contains 10 exons and 9 introns. It must be noted that the deletions and/or mutations which affect the gene and which accordingly are capable of giving rise to the ALD disease or its variant AMN, can be situated either in the exons or in the introns. When these modifications affect the intron sequences, they are often located in the sequence of the intron which is adjacent to the coding sequence. Presence of a mutation in the gene is a condition necessary for the expression of the disease but can remain insufficient to lead to the expression of clinical symptoms related to this disease.
  • the nucleotide sequence consists of the coding sequence of the gene.
  • this coding sequence can be a cDNA corresponding to the sequence represented on FIG. 2 (SEQ ID NO:1).
  • the isolated DNA sequence is characterized in that it consists essentially of a DNA sequence encoding the human adrenoleukodystrophy protein.
  • the DNA sequence codes for the amino-acid sequence represented on FIG. 2 (SEQ ID NO:2).
  • the invention further rebates to nucleotide sequences which are modified regarding the above described sequence but which nevertheless hybridize under stringent conditions defined hereafter with a nucleotide sequence as described above.
  • a sequence contains preferably at least 10 nucleotides and has advantageously a length of around 20 to 100, preferably 20 to 50 nucleotides.
  • the invention also concerns nucleotide fragments selected among DNA or cDNA fragments, which contain at least 10 nucleotides and advantageously at least 20 nucleotides, and are capable of hybridizing specifically, with a sequence which has been defined hereabove.
  • the hybridization is said specific if the nucleotide fragment does not hybridize for instance with the DNA sequence of the human PMP protein or the DNA of other proteins of the ATP binding protein superfamily.
  • the fragments of the invention can be labelled in order for instance to be used as probes or can be also involved as primers for amplification reactions and especially for PCR reactions.
  • the probes may be label led at the 3 ' end by addition of one or more deoxynucleotides or ribonucleotides or by a dideoxynucleotide labelled in the alpha position by means of 32 P, in the presence of the terminal deoxynucleotidyl transferase, or at the 5' end by transfer of a radioactive phosphate group of a free deoxynucleotide or dideoxynucleotide labelled in the gamma position in the presence of the T4 DNA ligase.
  • the probes may also be labelled by using a DNA polymerase by means of "nick translation” or "random priming" or "polymerase chain reaction".
  • the method of detection of the hybridization will depend on the radioactive label used and may be based on autoradiography, liquid scintillation, gamma counting or any other technique making possible the detection of the radiation emitted by the radioactive label.
  • Non-radioactive labelling may also be used by combining with probes groups exhibiting immunological properties such as an antigen, a specific affinity for certain reagents such as a ligand, physical properties such as fluorescence or luminescence, properties making possible the completion of enzymatic reactions such as an enzyme or an enzyme substrate.
  • the non-radioactive labelling may also be performed directly by chemical modification of the DNA, such as photobiotinylation or sulfonation.
  • Particularly preferred fragments to be used as primers for amplification procedures are those which are situated within the sensitive parts of the gene, i.e., the parts which may be more susceptible to mutations or deletions, or also fragments which are surrounding these regions.
  • interesting primers are for instance those corresponding to sequences extending from position 1,853 to position 1,872 or from position 1,854 to position, 1,874 or from position 2,357 to position 2,375 as shown on FIG. 2 (SEQ ID NO:1).
  • the diagnosis can be made to detect the anomaly of the gene in patients presenting clinical symptoms of the disease or unaffected persons capable of transmitting the disease, especially in women carriers or as a neonatal screening.
  • the invention at so concerns a pharmaceutical composition
  • a pharmaceutical composition comprising an isolated nucleotide sequence according to the definitions given above, together with a physiological acceptable pharmaceutical vehicle.
  • a particularly useful pharmaceutical composition is one which contains the genomic DNA of the ALD gene or the cDNA corresponding thereto.
  • Another aspect of the invention relates to a protein consisting essentially of the adrenoleukodystrophy protein.
  • this protein characterized by the amino-acid sequence given in FIG. 2 (SEQ ID NO:2).
  • the invention also relates to a protein having a sufficient homology with the amino-acid sequence given above, to have the essential biological properties the ALD protein.
  • These biological properties are the ability to complement the biological defect in cells from patients with ALD or AMN, or to share immunological determinants (epitopes) with the ALD protein.
  • Another protein within the scope of the invention has the essential biological properties described above and is such that the 52 amino-acid residues of its C-terminal end have an homology or at least 75%, preferably 80% and more preferably 90% with the aligned amino-acid sequence which has been given above.
  • the invention also relates to amino-acid fragments or sequences containing at least 7 amine-acid residues, which fragments are recognized by antibodies that bind specifically the protean of the invention.
  • Preferred fragments contain from 7 to around 745 or 500 amino-acids, advantageously from 7 to around 100, preferably from 7 to around 50 and most preferably from 7 to around 20.
  • Particular amino-acid sequences are derived from regions of the sequence represented on FIG. 2, which are specific for the ALD protein and especially which are not common to sequences of human and rat 70KPMP protein and other members of the ATP binding protein superfamily, such as those described in the publication of Mosser J. et al (Nature, vol. 361, 25 Feb. 1993, pages 736-730).
  • the antibodies capable of binding preferably specifically the ALD protein of the invention, especially monoclonal antibodies are obtained according to usual methods, involving the production of hybridoma cells, formed by fusion of spleen cells of an animal previously immunized with a protein of the invention and myeloma cells.
  • Specific monoclonal antibodies detect a protein having an apparent molecular weight of 75 kDa.
  • the invention further encompasses the antibodies, either monoclonal or polyclonal that bind specifically the protein according to the above definition.
  • the specific binding can be checked by assaying these antibodies with proteins having homologies with the ALD protein, for instance with the rat or human 70 peroxisomal membrane protein (PMP) or other proteins of the ATP binding protein superfamily as cited above.
  • the antibodies of the invention are those which do not bind these different proteins presenting some homology with the ALD protein.
  • nucleotide sequence or protein or fragments thereof of the invention are useful for diagnostic or therapeutic purposes.
  • nucleotide sequences or fragments as defined above can be used in a process for the in vitro diagnosis of the ALD or AMN disease in a human patient, these sequences being used as probes or as primers.
  • Usual techniques like those which are involved in the detection of genetic diseases are for instance southern blotting RFLP (Restriction Fragment Length, Polymorphism) detection or PCR reactions.
  • the detection of the protein can be performed using specific antibodies, monoclonal or polyclonal.
  • the detection is performed on samples containing for instance blood cells.
  • the invention also relates to a process for the treatment of cells and especially somatic cells of a human patient affected by ALD or AMN, comprising the administration to the patient of cells previously modified with a nucleotide sequence as described above.
  • the cells can be modified by recombinant nucleotide sequences containing one of the DNA, cDNA or RNA sequence of the invention, under the control of regulation elements in a vector appropriate for the modification or transfection of cells.
  • the regulation elements are capable of ensuring a high level of expression and comprise accordingly strong promoters, possibly an enhancer and in some instances a reporter gene such as for example the neo gene or the dhfr gene.
  • Appropriate vectors can be plasmid vectors, retroviruses vectors or for instance adenoviruses vectors.
  • the transfer of the sequence useful for therapeutic purposes is performed by ex vivo techniques like electroporation, transfection especially calcium phosphase transfection or fusion for instance with liposomes.
  • the somatic transfer can also be performed in vivo using cells as vectors, which cells are previously modified ex vivo with the gene of interest. Accordingly hematopoietic cells or nervous cells are used.
  • inert vectors like liposomes, viral vectors, especially retroviruses or adenoviruses or directly by injection of DNA.
  • the vectors used for the somatic transfer of the gene or sequences of the invention can also be directly transfered in vivo, for instance by direct injection in the blood stream or by stereotactic injection in specific regions of the brain (Strattford-Perricaudet LD et al J Clin Invest 90, 626-630. Akli Set al Nature genomics vol 3, March 1993).
  • the sequence of the ALD gene preferably the cDNA corresponding to the ALD gene is inserted in a defective murine Moloney vector (Mo-MLV), under the control of regulation elements.
  • the defective vector still contains its cis-sequences such as the LTR sequence or part thereof sufficient for the transcription and integration, the psi sequence necessary for the encapsidation and the PB sequence for viral replication.
  • the vital genes gag, pol, env are at least partly deleted and substituted with the sequence of interest. This sequence is placed under the control of its own promoter or a stronger promoter such as the SV40 promoter.
  • a marker gene is possibly added to the construction.
  • the helper virus used contains the retroviral genes (gag, pol, env) necessary for replication of the vital genome and for the formation of the vital particles. To the contrary the cis-sequences which are present in the vector are deleted in the helper virus.
  • the helper provirus is inserted in a murine cell line especially NIH/3T3 as host.
  • the vector is then transfected in the cell line allowing the production of viral particles.
  • the techniques used for the transfer of the human ADA gene (Adenosine denaminase) in cells can be also used in the present situation.
  • the ALD gene or its cDNA is transfected with a retrovirus in fibroblast cells (Palmer et al. PNAS, 1987, 84, 1055-1059), or in lymphocytes or other hematopoietic cells including proursor or stem cells (Culver et al. Hum Gene Ther 2,107 1991 or Anderson WF Science vol 256 May 6 1992 p808)
  • the joining of BP4-BP2 and BP3-BP1 were demonstrated by cloning the corresponding junction fragments (dashed lines).
  • the extends of the two deletions (19.2 and 88 kb respectively) are indicated in b.
  • Probe X2 (circle) is discussed in the examples and other probes (filled circles, Fr probes) are from Martinez, C. M. et al, (Cell Biol. int. Rep. 14, 255-266, 1990).
  • the distance between probes Fr14 and Fr11 (broken line) is not known.
  • the green cone pigment gene (GCP) is indicated by a box (G) .
  • Map of the R/GCP genes and of the second deletion in patient O including the rare cutter restriction sites (vertical bars, top) EagI (Ea), BssHII (Bs) , SacI (S), ClaI (C), NotI (N) and MluI (M).
  • the rare cutter sites within the pigment gene-repeat unit are not marked.
  • the extents of the two deletions (wavy lines) and the position of the 4 BPs in patient O are indicated.
  • Probe Fr15.4 (deleted in patient O) was used to screen a Xq28-specific cosmid library, yielding 3 overlapping clones (cos Qc 11H12, cos Qc 8F3 and cos Qc 14A11).
  • the red cone pigment (RCP) gene is indicated by the filed box (R) and the GCP bene by a box (G. c, Deletions detected in 5 ALD patients and restriction map of the subcloned region of ALD gene using the following enzymes: EcoRl (E), HindIII (H), BamHI (B), and XbaI (X). Rare cutter sites are indicated as in b. Localization or subcloned probes is shown at the top.
  • Probe X2 (box) is a 1.8 kb XbaI-EcoRT restriction fragment derived from X-8, the second junction fragment (BP4-BP2) in patient O.
  • X-8 was isolated from a XbaI genomic library constructed in bacteriophage (Stratagene) using DNA from a somatic hybrid line containing the X chromosome of patient O. Gel electrophoresis, southern blotting, probing and autoradiography were all done as described.
  • FIGS. 2A-2B Sequence of ALD-protein cDNA (SEQ ID NOS:1 and 2). The sequence is derived from analysis of clones obtained by exon connection and clones isolated from a HcLa cDNA library, and confirmed in most cases by sequences of genomic clones.
  • RESULTS RESULTS.
  • probes (Ta25, Ta1, Ta18 and TA13b) derived from subcloning of cosmid Qc 11H12 were sequenced.
  • Candidate exons, strand and frame assignments were screened by the GRAIL program (Oak Ridge, RN).
  • Oligonucleotide primers were designed according to the coding regions that presented highest homology score to 70K PMP in TA25, TA18, and TA13b.
  • Ex13 and Ex3 are cDNA clones obtained by exon connection in 2-step-boosted or nested polymerase reactions (30 cycles with external primers and 40 cycles with internal primers) performed on oligo(dT)-primed cDNA Total RNA (20 ⁇ g) from a lymphoblastoid cell line was used as starting material.
  • External primers correspond to positions 1,853-1,872 (for Ex13), and to positions 1,854-1,874 and 2,357-2,375 (for Ex3).
  • Internal primers are indicated by arrows.
  • FIGS. 3a-3b Detection of deletions in the DNA isolated from three ALD patients using cDNA probe Ex13.
  • DNA was digested with EcoRI (lates 1-4), HindIII (lanes 5-8) and TaqI (lanes 9-12).
  • Lanes 1, 5 and 9 normal woman; lanes 2, 6 and 10: patient L; lanes 3, 7 and 11: patient R; lanes 4, 8 and 12: patient Ma.
  • the 10.8-kb EcoRI normal fragment (lanes 2-4 ) and the 12.5 -kb HindIII abnormal junction fragment from patient R (lane 7) hybridize to only around 60 bp of the Ex13 probe and are thus very fait. Sizen (in kb) of normal restriction fragments are indicated on the left and on the right.
  • RNA size markers are indicated on the left.
  • a human, ⁇ -actin probe hybridized to the same northern blot detects two transcripts of 2.0 and 1.8 kb, respectively.
  • METHODS Gel electrophoresis, southern blotting, probing and autoradiography were done as described.
  • human multiple-tissue northern blot was purchased from Clontech. Membranes were exposed at -70° C. to X-ray film for 5d (Ex13 or EX3 probes) or for 6 h ( ⁇ -actin probe).
  • FIGS. 4A-4B Sequence alignment of ALD protein (SEQ ID NO:2) and human 70K PMP (SEQ ID NO:3). Amino acid identities are indicated by two dots and conservative changes by one. Sequence similarities were established with the FASTP program.
  • FIG. 5. Structural organization of the ALD gene The distribution of the 10 ALD exons is shown with black boxes. Traduction initiation and termination sites (respectively in exon 1 and 10) are indicated. The location of the CpG island, the genomic probe FR15.4 (grey box) and the most centrometric breakpoint of patient O rearrangement (BP4) are also represented.
  • FIGS. 7A-7D Complete DNA sequence of the ALD gene (SEQ ID NO:18-23). It contains both the cDNA sequence (in capital letters) and the intron sequence (in small letters).
  • Probes cot-responding to three of the breakpoints (BP) of this rearrangement were isolated (BP1, BP2 and BP3) (FIG. 1).
  • BP breakpoints
  • BP3 BP3
  • a probe 4 kb proximal to Fr15 was used (deleted in patient O; Martinez et al, Cell Biol. Int. Rep. 14,255-266 (1990) to obtain clones from a Xq28 cosmid library.
  • Three overlapping clones were obtained that spanned about 90 kb and contained a cluster of rare restriction cutting sites (EagI, BssHII, SacII) indicating the presence of a CpG island (FIG. 1c).
  • XbaI junction fragment (X-8) corresponding to breakpoints 4 and 2 in patient O was cloned (FIG. 1a) .
  • a restriction map of X-8 showed that a 1-8 kb XbaI-EcoRI (X2) fragment contains a 1.6-kb segment on the BP4 side and included within the Fr15 cosmid contig described above (FIG. 1b and c). Breakpoints 3 and 4 are separated by 19.2 kb, and delimit the second deletion in patient O.
  • probe TA25, TA1, TA18 and TA13B (FIG. 1c) showed cross-hybridization to various mammalian species. More important, X2, TA4, TA18 and Ta13a probes detected deletions in five other ALD patients. Probe X2 allowed the detection of a junction fragment segregating with the disease in family B (FIG. 1d). In another family (Ma), probe X2 detected an identical 22-kh HindIII junction fragment in two brothers with different clinical phenotypes of ALD (FIG. 1e).
  • deletions ranged from 1.6 kb (patient B) to 15.3 kb (patient R) with partial overlap (FIG. 1c).
  • Six patients had deletions (including patient O) in a population of 85 independent ALD patients, but no deletions were found in a panel of 82 control males. These results indicate that the region contains at least part or the ALD gene.
  • the sequences of probes Ta25, TA18, TA1.3b and of TA13a were determined and examined for putative coding regions (using a computer program based on a multiple sensor-neural netword approach) revealed a large (>700 bp) putative protein coding sequence within TA25 and smaller open reading frames (300-400 bp) in TA18, TA13 a and TA13b.
  • the deduced amino-acid sequences showed significant sequence identity with collinearly positioned regions of human or rat 70K peroxisomal membrane protein (PMP). Nested-PCR reactions using primers from the putative exons (FIG.
  • Ex13 and Ex3 produced two fragments (Ex13 and Ex3) of sizes (645 bp and 498 bp, respectively) compatible with those expected from homology with the 70K PMP cDNA. They hybridized to the predicted DNA fragments in normal individuals and to the same fragments detected by genomic probes in ALD patients (FIG. 3a). Ex13 and Ex3 have been used in combination to screen a random-primed HeLa cell cDNA library to obtain 6 independent overlapping clones.
  • the 2,751-bp sequence (SEQ ID NO:2) contains the whole protein-coding sequence of 745 amino acids (SEQ ID NO:2).
  • the first methionine codon is preceded by an in-phase stop codon (at bp 282) and is included within a potential ribosome-binding sequence.
  • Significant sequence identity with the 70K PMP (SEQ ID NO:3) begins at methionine 67 and ends at around 680 (corresponding to the carboxy terminus of 70K PMP).
  • the remaining 52 amino acid residues are unique to the ALD protein (FIG. 4a).
  • ALD protein (SEQ ID NO:2) could be aligned with human 70K PMP (SEQ ID NO:3) (Saari, J. C. & Bredberg, L. Biochim. biophys. Acta 716, 266-272, 1982) ) with only a few deletions or insertions, and revealed a 38.5% amino-acid identity (253/659 amino acids) (FIG. 4a).
  • SEQ ID NO:3 human 70K PMP
  • FIG. 4a The sequence similarity between the two protein sequences reached 78.9%.
  • the two proteins show a similar hydrophobicity profiles, with a hydrophobic amino-terminal region containing potential transmembrane segments.
  • the hydrophilic carboxy-terminal region of the ALD protein shows 56% identity over 210 amino acids to the corresponding region of 70K PMP.
  • the two characteristic nucleotide-binding consensus sequences are almost identical between the two proteins (underlined in FIG. 4b).
  • a transcript of 4,2 kb was detected in heart, placenta, lung, liver, skeletal muscle, testis, pancreas and, to a lesser extent, in brain and kidney (FIG. 3b).
  • the expression of the 4.2-kb transcript was very low in adult brain but more marked in 21-week fetal brain.
  • a second transcript of 6.8 kb was detected in heart and skeletal muscle, whereas a third transcript of 2.75 kb was detected in muscle and liver.
  • the putative ALD gene has thus been identified in the distal part of Xq28 which has deletions in one or several exons in 6 of 85 independent ALD patients. Some of these deletions are small and non-overlapping, thus strengthening the conclusion that this gene is indeed involved in ALD.
  • the gene coding for VLCFA-CoA synthetase was considered as a candidate gene for ALD, a recently cloned rat gene for long-chain (C 12 -C 16 ) acyl-CoA synthetase failed to detect homologous sequences on the X chromosome.
  • the putative ALD gene shows no homology to this latter sequence, or to the three other enzymes involved in peroxisomal ⁇ -oxidation.
  • the conserved region includes the A and B consensus sequences (A; G-X4-G-K-T-X6-I/V; B: R/K-X3-G-X3-L(hydrophobic)4-D) of a nucleotide-binding fold, plus a 12-amino-acid motif highly conserved in ABC proteins (FIG. 4b).
  • ALD protein must therefore be a member of this superfamily of ABC transporters, which are also involved in transport of proteins, amino acids, inorganic ions and peptides in prokaryotes and eukaryotes.
  • SEQ ID NO:2 shows significant identity to 70K PMP (SEQ ID NO:3), no homology was found to 35K PMP or to other PMPs required for peroxisome biogenesis in yeast.
  • ALD was initially thought to involve a deficiency in peroxisomal VLCFA-CoA synthesase
  • the predicted sequence of the putative ALD protein rather suggests a protein involved in transport of VLCFA-CoA synthetase into the peroxisomal membrane or a protein that is functionally associated with the VLCFA-CoA synthetase in the peroxisomal membrane.
  • the translocation of acyl-CoA oxidase, the next enzyme or the peroxisomal ⁇ -oxidation pathway, requires ATP hydrolysis, whereas the transport of VLCFA across the peroxisomal membrane does not, and neither is it impaired in peroxisomes from ALD patients.
  • ALD protein was observed in every tissue tested, but the relationship between ALD protein expression and the abundance of peroxisomes in tissues may not be straight-forward. Perosisomes are particularly abundant in liver and kidney, having an average diameter of 0.2-1 ⁇ M. In other tissues, including the brain and fibroblasts, they are less abundant and smaller (0.05-0.2 ⁇ M). This abundance and size difference may reflect a distinct membrane and matrix protein compositions of peroxisomes in different tissues. Although ALD is associated with a defective oxidation of VLCFA, this metabolic defect is mainly expressed in brain and adrenal tissues.
  • ALD is characterized by a striking variation in clinical phenotype.
  • family Ma an identical deletion was found in two sibs, a boy who developed cerebral ALD at 8 years, and his brother who developed only very mild adrenal insufficiency at 13 years.
  • deletions were associated with the adult form (patients O and R) as well as with the severe childhood form (patients B and L).
  • a defective murine Moloney vector (Mo-MLV) is used.
  • the defective vector still contains its cis-sequences such as the LTR sequence or part the thereof sufficient for the transcription and integration, the psi sequence necessary for the encapsidation and the PB sequence for Vital replication.
  • the viral genes gag, pol, env are at least partly deleted.
  • the promoter of the ALD gene or advantageously a stronger promoter such as the PGK-1 promoter (phosphoglycerate kinase) or SV10 promoter replace the deleted vital gene.
  • the CDNA of the ALD gene is cloned within the defective vector, under the control of the promoter, in a chosen restriction site.
  • the retroviral vector is then introduced in a cell line for encapsidation, which cell line expresses the gag, pol and env vital genes.
  • a cell line like NIH/3T3 previously modified with the helper virus (Danos et al, PNAS, 85, 6460-6465, 1988) is used.
  • the recombinant construct is introduced by transfection and the cells produce vital particles.
  • the cells used for the transfer of the cDNA sequence are cultivated and contacted and incubated with the retroviral vector.
  • the infected cells are then amplified sufficiently to be used for the treatment.

Abstract

The cDNA and genomic DNA sequences encoding the human adrenoleukodystrophy protein are provided. Mutations of the adrenoleukodystrophy protein cause adrenoleukodystrophy or adrenomyelopathy.

Description

BACKGROUND OF THE INVENTION
The present application relates to the identification and isolation of a gene which is responsible for the adrenoleukodystrophy. It further concerns the protein encoded by this gene and their use in diagnostic or therapeutic procedures.
Adrenoleukodystrophy (ALD) is an X-linked disease affecting 1/20,000 males either as cerebral ALD in childhood or as adrenomyeloueuropathy (AMN) in adults. Childhood ALD is the more severe form, with onset of neurological symptoms between 5-12 years of age. Central nervous system demyelination progresses rapidly and death occurs within a few years. AMN is a milder form of the disease with onset at 15-30 years of age and a more progressive course. Adrenal insufficiency (Addison's disease) may remain the only clinical manifestation of ALD. The principal biochemical abnormality of ALD is the accumulation of very long chain fatty acids (VLCFA) because of impaired β-oxidation in peroxisomes. The normal oxidation of VLCFA-CoA in patients fibroblasts suggested that the gene coding for the VLCFA-CoA synthetase could be a candidate gene for ALD.
ALD or its variant ANN is a monogenic disease but its clinical expression can be under the control of several genes or factors, leading to phenotypic variability.
Adrenoleukodystrophy and adrenomyeloneuropathy are characterized by the presence of an abnormal ALD gene, resulting from deletions or other types of mutations including point mutations. The mututions in the gene may nevertheless remain clinically silent or may lead to various phenotypic clinical expression.
Although it well known that the gene responsible for the adrenoleukodystrophy is located on the Xq28 region of the X chromosome, the results which have been described up to now have not permitted to identify and characterize the gene responsible for the ALD.
Some experiments were for instance conducted in order to check any possible relationship between the alteration of the gene responsible for the colour vision and the ALD gene. The inventors have now shown that although these genes of the red/green colour pigment also map to the Xq28 region, they are not linked either structurally or functionally to the ALD gene.
For the purpose of this description, it is mentioned that the expression "ALD gene" encompasses the gene involved in ALD and also in its adult variant AMN.
SUMMARY OF THE INVENTION
The invention accordingly relates to an isolated nucleotide sequence which is for instance selected among DNA, RNA, CDNA sequences, responsible for the adrenoleukodystrophy or the adrenomyeloneuropathy.
By the expression "sequences responsible for ALD or AMN" it must be understood that the abnormal form of the ALD gene is involved in the ALD pathology; of course the normal gene (devoid of mutations, especially of deletions) does not cause the disease.
The term "isolation" refers to the fact that the nucleotide sequence is separated from the other nucleotide sequences of the Xq28 region of the X chromosome when it is purified for instance from a natural source or organism. This isolated nucleotide sequence is also obtainable from synthetic or semi-synthetic sources, according to well-known methods. This sequence can be any type of nucleotide sequence and especially can be selected among DNA, RNA or cDNA.
A particular sequence which is referred to according to the invention consists essentially of the human gene responsible for the ALD. This gene accordingly contains both exons and introns and therefore contains both coding sequences of the gene and regulation sequences.
A preferred embodiment of the invention provides for an isolated nucleotide sequence having the sequence represented on FIG. 6 (SEQ ID NO:4-17) and FIG. 7 (SEQ ID NO:8-23).
The inventors have shown that the gene coding for the ALD protein, contains 10 exons and 9 introns. It must be noted that the deletions and/or mutations which affect the gene and which accordingly are capable of giving rise to the ALD disease or its variant AMN, can be situated either in the exons or in the introns. When these modifications affect the intron sequences, they are often located in the sequence of the intron which is adjacent to the coding sequence. Presence of a mutation in the gene is a condition necessary for the expression of the disease but can remain insufficient to lead to the expression of clinical symptoms related to this disease.
According to another preferred embodiment of the invention, the nucleotide sequence consists of the coding sequence of the gene.
In particular, this coding sequence can be a cDNA corresponding to the sequence represented on FIG. 2 (SEQ ID NO:1).
According to another embodiment of the invention, the isolated DNA sequence is characterized in that it consists essentially of a DNA sequence encoding the human adrenoleukodystrophy protein. In such a case, the DNA sequence codes for the amino-acid sequence represented on FIG. 2 (SEQ ID NO:2).
The invention further rebates to nucleotide sequences which are modified regarding the above described sequence but which nevertheless hybridize under stringent conditions defined hereafter with a nucleotide sequence as described above. Such a sequence contains preferably at least 10 nucleotides and has advantageously a length of around 20 to 100, preferably 20 to 50 nucleotides.
A preferred nucleotide sequence of the invention can further be characterized by the restriction map which is given on FIG. 5 or by the structural organization which is also given on FIG. 5.
The invention also concerns nucleotide fragments selected among DNA or cDNA fragments, which contain at least 10 nucleotides and advantageously at least 20 nucleotides, and are capable of hybridizing specifically, with a sequence which has been defined hereabove.
The hybridization is said specific if the nucleotide fragment does not hybridize for instance with the DNA sequence of the human PMP protein or the DNA of other proteins of the ATP binding protein superfamily.
The fragments of the invention can be labelled in order for instance to be used as probes or can be also involved as primers for amplification reactions and especially for PCR reactions.
The probes of the invention are advantageously labelled by any label classically used. They may be labelled with the aid of a radioactive marker such as 32 P, 35 S, 125 I, 3 H, 14 C and the radioactive labelling may be performed by any method known to the person skilled in the art.
The probes may be label led at the 3 ' end by addition of one or more deoxynucleotides or ribonucleotides or by a dideoxynucleotide labelled in the alpha position by means of 32 P, in the presence of the terminal deoxynucleotidyl transferase, or at the 5' end by transfer of a radioactive phosphate group of a free deoxynucleotide or dideoxynucleotide labelled in the gamma position in the presence of the T4 DNA ligase. The probes may also be labelled by using a DNA polymerase by means of "nick translation" or "random priming" or "polymerase chain reaction".
The method of detection of the hybridization will depend on the radioactive label used and may be based on autoradiography, liquid scintillation, gamma counting or any other technique making possible the detection of the radiation emitted by the radioactive label.
Non-radioactive labelling may also be used by combining with probes groups exhibiting immunological properties such as an antigen, a specific affinity for certain reagents such as a ligand, physical properties such as fluorescence or luminescence, properties making possible the completion of enzymatic reactions such as an enzyme or an enzyme substrate. The non-radioactive labelling may also be performed directly by chemical modification of the DNA, such as photobiotinylation or sulfonation.
A particular fragment which can be used as a probe is the fragment which is designated by "X2" and which corresponds to an XbaI-EcoRI fragment of 1,8 kb included in the sequence represented on FIG. 6.
Other preferred probes are those designated by Ex13 and Ex3 and represented on FIG. 2.
Particularly preferred fragments to be used as primers for amplification procedures are those which are situated within the sensitive parts of the gene, i.e., the parts which may be more susceptible to mutations or deletions, or also fragments which are surrounding these regions.
Interesting primers are for instance those corresponding to sequences extending from position 1,853 to position 1,872 or from position 1,854 to position, 1,874 or from position 2,357 to position 2,375 as shown on FIG. 2 (SEQ ID NO:1).
The diagnosis can be made to detect the anomaly of the gene in patients presenting clinical symptoms of the disease or unaffected persons capable of transmitting the disease, especially in women carriers or as a neonatal screening.
The invention at so concerns a pharmaceutical composition comprising an isolated nucleotide sequence according to the definitions given above, together with a physiological acceptable pharmaceutical vehicle.
A particularly useful pharmaceutical composition is one which contains the genomic DNA of the ALD gene or the cDNA corresponding thereto.
Another aspect of the invention relates to a protein consisting essentially of the adrenoleukodystrophy protein.
In a preferred embodiment, this protein characterized by the amino-acid sequence given in FIG. 2 (SEQ ID NO:2).
The invention also relates to a protein having a sufficient homology with the amino-acid sequence given above, to have the essential biological properties the ALD protein.
These biological properties are the ability to complement the biological defect in cells from patients with ALD or AMN, or to share immunological determinants (epitopes) with the ALD protein.
Another protein within the scope of the invention has the essential biological properties described above and is such that the 52 amino-acid residues of its C-terminal end have an homology or at least 75%, preferably 80% and more preferably 90% with the aligned amino-acid sequence which has been given above.
The invention also relates to amino-acid fragments or sequences containing at least 7 amine-acid residues, which fragments are recognized by antibodies that bind specifically the protean of the invention.
Preferred fragments contain from 7 to around 745 or 500 amino-acids, advantageously from 7 to around 100, preferably from 7 to around 50 and most preferably from 7 to around 20.
Particular amino-acid sequences are derived from regions of the sequence represented on FIG. 2, which are specific for the ALD protein and especially which are not common to sequences of human and rat 70KPMP protein and other members of the ATP binding protein superfamily, such as those described in the publication of Mosser J. et al (Nature, vol. 361, 25 Feb. 1993, pages 736-730).
The antibodies capable of binding preferably specifically the ALD protein of the invention, especially monoclonal antibodies are obtained according to usual methods, involving the production of hybridoma cells, formed by fusion of spleen cells of an animal previously immunized with a protein of the invention and myeloma cells.
Specific monoclonal antibodies detect a protein having an apparent molecular weight of 75 kDa.
The invention further encompasses the antibodies, either monoclonal or polyclonal that bind specifically the protein according to the above definition.
The specific binding can be checked by assaying these antibodies with proteins having homologies with the ALD protein, for instance with the rat or human 70 peroxisomal membrane protein (PMP) or other proteins of the ATP binding protein superfamily as cited above. The antibodies of the invention are those which do not bind these different proteins presenting some homology with the ALD protein.
The nucleotide sequence or protein or fragments thereof of the invention are useful for diagnostic or therapeutic purposes.
Especially the nucleotide sequences or fragments as defined above can be used in a process for the in vitro diagnosis of the ALD or AMN disease in a human patient, these sequences being used as probes or as primers.
Usual techniques like those which are involved in the detection of genetic diseases are for instance southern blotting RFLP (Restriction Fragment Length, Polymorphism) detection or PCR reactions.
The detection of the protein can be performed using specific antibodies, monoclonal or polyclonal.
The detection is performed on samples containing for instance blood cells.
In the present case due to the possibility of various mutations of the ALD gene, it can be useful to have recourse to multiplex PCR, using different primers for amplification of several regions of the gene.
The invention also relates to a process for the treatment of cells and especially somatic cells of a human patient affected by ALD or AMN, comprising the administration to the patient of cells previously modified with a nucleotide sequence as described above. The cells can be modified by recombinant nucleotide sequences containing one of the DNA, cDNA or RNA sequence of the invention, under the control of regulation elements in a vector appropriate for the modification or transfection of cells. Advantageously the regulation elements are capable of ensuring a high level of expression and comprise accordingly strong promoters, possibly an enhancer and in some instances a reporter gene such as for example the neo gene or the dhfr gene.
Appropriate vectors can be plasmid vectors, retroviruses vectors or for instance adenoviruses vectors.
The transfer of the sequence useful for therapeutic purposes is performed by ex vivo techniques like electroporation, transfection especially calcium phosphase transfection or fusion for instance with liposomes.
The somatic transfer can also be performed in vivo using cells as vectors, which cells are previously modified ex vivo with the gene of interest. Accordingly hematopoietic cells or nervous cells are used. Among the techniques which are available for in vivo transfer of gene one can further cite inert vectors like liposomes, viral vectors, especially retroviruses or adenoviruses or directly by injection of DNA.
The vectors used for the somatic transfer of the gene or sequences of the invention can also be directly transfered in vivo, for instance by direct injection in the blood stream or by stereotactic injection in specific regions of the brain (Strattford-Perricaudet LD et al J Clin Invest 90, 626-630. Akli Set al Nature genomics vol 3, March 1993).
The direct administration of DNA has been described for instance by Wolff J. A. et al (1990, Science 247, 1465-1468) or Acsadi G. et al (1991, Nature 352, 815-818).
As example, for the preparation, of the vector, the sequence of the ALD gene, preferably the cDNA corresponding to the ALD gene is inserted in a defective murine Moloney vector (Mo-MLV), under the control of regulation elements. The defective vector still contains its cis-sequences such as the LTR sequence or part thereof sufficient for the transcription and integration, the psi sequence necessary for the encapsidation and the PB sequence for viral replication. In this vector the vital genes gag, pol, env are at least partly deleted and substituted with the sequence of interest. This sequence is placed under the control of its own promoter or a stronger promoter such as the SV40 promoter. A marker gene is possibly added to the construction.
The helper virus used contains the retroviral genes (gag, pol, env) necessary for replication of the vital genome and for the formation of the vital particles. To the contrary the cis-sequences which are present in the vector are deleted in the helper virus.
The helper provirus is inserted in a murine cell line especially NIH/3T3 as host.
The vector is then transfected in the cell line allowing the production of viral particles.
From a general point of view, the techniques used for the transfer of the human ADA gene (Adenosine denaminase) in cells can be also used in the present situation. Like the ADA gene, the ALD gene or its cDNA is transfected with a retrovirus in fibroblast cells (Palmer et al. PNAS, 1987, 84, 1055-1059), or in lymphocytes or other hematopoietic cells including proursor or stem cells (Culver et al. Hum Gene Ther 2,107 1991 or Anderson WF Science vol 256 May 6 1992 p808)
Other characteristics and advantages of the invention will become apparent from the examples add figures.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG 1a-1e Map of the ALD gene region and its rearrangements in patients a, Chromosomal rearrangement in patient O. The joining of BP4-BP2 and BP3-BP1 were demonstrated by cloning the corresponding junction fragments (dashed lines). The extends of the two deletions (19.2 and 88 kb respectively) are indicated in b. Probe X2 (circle) is discussed in the examples and other probes (filled circles, Fr probes) are from Martinez, C. M. et al, (Cell Biol. int. Rep. 14, 255-266, 1990). The distance between probes Fr14 and Fr11 (broken line) is not known. The green cone pigment gene (GCP) is indicated by a box (G) . b, Map of the R/GCP genes and of the second deletion in patient O, including the rare cutter restriction sites (vertical bars, top) EagI (Ea), BssHII (Bs) , SacI (S), ClaI (C), NotI (N) and MluI (M). The rare cutter sites within the pigment gene-repeat unit are not marked. The extents of the two deletions (wavy lines) and the position of the 4 BPs in patient O are indicated. Probe Fr15.4 (deleted in patient O) was used to screen a Xq28-specific cosmid library, yielding 3 overlapping clones (cos Qc 11H12, cos Qc 8F3 and cos Qc 14A11). The red cone pigment (RCP) gene is indicated by the filed box (R) and the GCP bene by a box (G. c, Deletions detected in 5 ALD patients and restriction map of the subcloned region of ALD gene using the following enzymes: EcoRl (E), HindIII (H), BamHI (B), and XbaI (X). Rare cutter sites are indicated as in b. Localization or subcloned probes is shown at the top. Probe X2 (box) is a 1.8 kb XbaI-EcoRT restriction fragment derived from X-8, the second junction fragment (BP4-BP2) in patient O. TA25 (2.1 kb), Ta4 (3.6 kb), Ta1 (1.0 kb), Ta18 (0.85 kb), Ta13a (935 bp) and Ta13b (252 bp) are TaqI-digested DNA fragments (hatched boxes) derived from subcloning of cosmid Qc 11H12. d, Segregation of an abnormal junction fragment detected by probe X2 in ALD family B. Probe X2 hybridizes to a 16-kb HindIII fragment in normal individuals (open square and circle). An abnormal 14.4-kb junction fragment was detected in an affected ALD patient (pat lent B; see panel c) and in all heterozygous females. e, Detection of the same rearranged DNA fragment in two ALD brothers with different clinical ALD phenotypes by southern blot analysis of HindIII-digested DNA from 3 brothers (Family Ma; sec c) hybridized with probe X2. An abnormal junction, fragment of 22kb is detected by X2 in a male with cerebral ALD (filled square) and his brother with Addison's disease (hatched square). METHODS. Restriction patterns of the cosmids were analysed as described. Cosmid Qc 11H12 was digested with TaqI and cloned directly in the ClaI site of pBluescript SK+ (Stratagene). X-8 was isolated from a XbaI genomic library constructed in bacteriophage (Stratagene) using DNA from a somatic hybrid line containing the X chromosome of patient O. Gel electrophoresis, southern blotting, probing and autoradiography were all done as described.
FIGS. 2A-2B: Sequence of ALD-protein cDNA (SEQ ID NOS:1 and 2). The sequence is derived from analysis of clones obtained by exon connection and clones isolated from a HcLa cDNA library, and confirmed in most cases by sequences of genomic clones.
METHODS. Four probes (Ta25, Ta1, Ta18 and TA13b) derived from subcloning of cosmid Qc 11H12 were sequenced. Candidate exons, strand and frame assignments were screened by the GRAIL program (Oak Ridge, RN). Oligonucleotide primers were designed according to the coding regions that presented highest homology score to 70K PMP in TA25, TA18, and TA13b. Ex13 and Ex3 are cDNA clones obtained by exon connection in 2-step-boosted or nested polymerase reactions (30 cycles with external primers and 40 cycles with internal primers) performed on oligo(dT)-primed cDNA Total RNA (20 μg) from a lymphoblastoid cell line was used as starting material. External primers correspond to positions 1,853-1,872 (for Ex13), and to positions 1,854-1,874 and 2,357-2,375 (for Ex3). Internal primers are indicated by arrows. Subsequent amplification products were blunt-ended by action or T4 DNA polymerase (New England Biolabs), directly cloned in pBlue-script KS+ vector (Stratagene), sequenced using dideoxynucleotide termination (applied Biosystems), and analysed on an automated DNA sequencer (Applied Biosystems).
FIGS. 3a-3b, Detection of deletions in the DNA isolated from three ALD patients using cDNA probe Ex13. DNA was digested with EcoRI (lates 1-4), HindIII (lanes 5-8) and TaqI (lanes 9-12). Lanes 1, 5 and 9: normal woman; lanes 2, 6 and 10: patient L; lanes 3, 7 and 11: patient R; lanes 4, 8 and 12: patient Ma. The 10.8-kb EcoRI normal fragment (lanes 2-4 ) and the 12.5 -kb HindIII abnormal junction fragment from patient R (lane 7) hybridize to only around 60 bp of the Ex13 probe and are thus very fait. Sizen (in kb) of normal restriction fragments are indicated on the left and on the right. Similar results were obtained with Ex3 in ALD patients deleted in 3' end region of the ALD gene. b, Northern blot analysis with probe ex13. cDNA Ex13 hybridized to northern blot (top) of human poly (A)+ RNA detects a transcript of 4.2 kb which is expressed in heart (H), placenta (P), lung (Lu), liver (Li) skeletal muscle (M), pancreas (Pa) and, to a lesser extent, in brain (B) and kidney (K). Two other transcripts are detected in heart and skeletal muscle (6.8 kb) and in liver and skeletal muscle (2.75 kb). RNA size markers are indicated on the left. A human, β-actin probe hybridized to the same northern blot (bottom panel) detects two transcripts of 2.0 and 1.8 kb, respectively. METHODS. Gel electrophoresis, southern blotting, probing and autoradiography were done as described. For b, human multiple-tissue northern blot was purchased from Clontech. Membranes were exposed at -70° C. to X-ray film for 5d (Ex13 or EX3 probes) or for 6 h (β-actin probe).
FIGS. 4A-4B. Sequence alignment of ALD protein (SEQ ID NO:2) and human 70K PMP (SEQ ID NO:3). Amino acid identities are indicated by two dots and conservative changes by one. Sequence similarities were established with the FASTP program. FIG. 5. Structural organization of the ALD gene The distribution of the 10 ALD exons is shown with black boxes. Traduction initiation and termination sites (respectively in exon 1 and 10) are indicated. The location of the CpG island, the genomic probe FR15.4 (grey box) and the most centrometric breakpoint of patient O rearrangement (BP4) are also represented. FIG. 6. Intron-exon boundaries of the ALD gene (SEQ ID NO.4-17). First and last two bases of each exon are indicated in bold. The position (number between brackets) correspond to the published cDNA sequence (Mosser et al, Nature). The average size of the gap within the sequence is specified for large introns. Sequence of small introns is fully represented.
FIGS. 7A-7D Complete DNA sequence of the ALD gene (SEQ ID NO:18-23). It contains both the cDNA sequence (in capital letters) and the intron sequence (in small letters).
DETAILED DESCRIPTION OF THE INVENTION EXAMPLES
I) Isolation and identification of the ALD gene
Here a positional cloning was used to identify a gene partially deleted in 6 of 85 independent patients with ALD. In familial cases, the deletions segregated with the disease. An identical deletion was detected in two brothers presenting with different clinical ALD phenotypes. Candidate exons were identified by computer analysis of genomic sequences and used to isolate complementary DNAs by exert connection and screening of cDNA libraries. The deduced protein sequence shows significant sequence identity to a peroxisomal membrane protein or Mr 70K that is involved in peroxisome biogenesis and belongs to the "ATP-binding cassette" superfamily of transporters.
As previous attempts to purify VLCF-CoA synthetase were unsucessful, a "positional cloning" approach was used. The ALD locus has been mapped to Xq28 (deDuve, C. et al, Biochem. Pharmac. 23, 2495-2531, 1974), where the red/green colour pigment (R/GCP) genes reside. On the basis of the high incidence (40%) of colour vision anomalies in AMN patients and earlier results, it was firstly proposed that ALD and R/GCP genes could be close together. Recently, an AMN patient with blue-monochromatic colour vision was identified who had a complex rearrangement located 5' of the red-colour pigment (RCP) gene, which included two deletions separated by a large (>110 kb) inversion (FIG. 1a and b). Only the RCP gene was found in the first deletion (88 kb). No additional deletion was detected in this region in 81 other ALD patients. It was then postulated that the inverted segment or the second deletion were candidate loci for the ALD gene.
Probes cot-responding to three of the breakpoints (BP) of this rearrangement were isolated (BP1, BP2 and BP3) (FIG. 1). To estimate the size of the second deletion a probe 4 kb proximal to Fr15 was used (deleted in patient O; Martinez et al, Cell Biol. Int. Rep. 14,255-266 (1990) to obtain clones from a Xq28 cosmid library. Three overlapping clones were obtained that spanned about 90 kb and contained a cluster of rare restriction cutting sites (EagI, BssHII, SacII) indicating the presence of a CpG island (FIG. 1c). In parallel, an XbaI junction fragment (X-8) corresponding to breakpoints 4 and 2 in patient O was cloned (FIG. 1a) . A restriction map of X-8 showed that a 1-8 kb XbaI-EcoRI (X2) fragment contains a 1.6-kb segment on the BP4 side and included within the Fr15 cosmid contig described above (FIG. 1b and c). Breakpoints 3 and 4 are separated by 19.2 kb, and delimit the second deletion in patient O.
To search for conserved sequences in other mammalian species and to detect additional deletions in other ALD patients, cosmid Qc 11H12 was subcloned (FIG. 1b, c). Probes TA25, TA1, TA18 and TA13B (FIG. 1c) showed cross-hybridization to various mammalian species. More important, X2, TA4, TA18 and Ta13a probes detected deletions in five other ALD patients. Probe X2 allowed the detection of a junction fragment segregating with the disease in family B (FIG. 1d). In another family (Ma), probe X2 detected an identical 22-kh HindIII junction fragment in two brothers with different clinical phenotypes of ALD (FIG. 1e). The size of deletions ranged from 1.6 kb (patient B) to 15.3 kb (patient R) with partial overlap (FIG. 1c). Six patients had deletions (including patient O) in a population of 85 independent ALD patients, but no deletions were found in a panel of 82 control males. These results indicate that the region contains at least part or the ALD gene.
The sequences of probes Ta25, TA18, TA1.3b and of TA13a were determined and examined for putative coding regions (using a computer program based on a multiple sensor-neural netword approach) revealed a large (>700 bp) putative protein coding sequence within TA25 and smaller open reading frames (300-400 bp) in TA18, TA13 a and TA13b. The deduced amino-acid sequences showed significant sequence identity with collinearly positioned regions of human or rat 70K peroxisomal membrane protein (PMP). Nested-PCR reactions using primers from the putative exons (FIG. 2) produced two fragments (Ex13 and Ex3) of sizes (645 bp and 498 bp, respectively) compatible with those expected from homology with the 70K PMP cDNA. They hybridized to the predicted DNA fragments in normal individuals and to the same fragments detected by genomic probes in ALD patients (FIG. 3a). Ex13 and Ex3 have been used in combination to screen a random-primed HeLa cell cDNA library to obtain 6 independent overlapping clones.
The 2,751-bp sequence (FIG. 2) (SEQ ID NO:2) contains the whole protein-coding sequence of 745 amino acids (SEQ ID NO:2). The first methionine codon is preceded by an in-phase stop codon (at bp 282) and is included within a potential ribosome-binding sequence. Significant sequence identity with the 70K PMP (SEQ ID NO:3) begins at methionine 67 and ends at around 680 (corresponding to the carboxy terminus of 70K PMP). The remaining 52 amino acid residues are unique to the ALD protein (FIG. 4a).
The sequence of ALD protein (SEQ ID NO:2) could be aligned with human 70K PMP (SEQ ID NO:3) (Saari, J. C. & Bredberg, L. Biochim. biophys. Acta 716, 266-272, 1982) ) with only a few deletions or insertions, and revealed a 38.5% amino-acid identity (253/659 amino acids) (FIG. 4a). When conservative amino acids substitutions are considered, the sequence similarity between the two protein sequences reached 78.9%. The two proteins show a similar hydrophobicity profiles, with a hydrophobic amino-terminal region containing potential transmembrane segments.
The hydrophilic carboxy-terminal region of the ALD protein shows 56% identity over 210 amino acids to the corresponding region of 70K PMP. The two characteristic nucleotide-binding consensus sequences are almost identical between the two proteins (underlined in FIG. 4b).
When northern blots of poly(A)+ RNAs from human tissues were hybridized to probes Ex13 or EX3, a transcript of 4,2 kb was detected in heart, placenta, lung, liver, skeletal muscle, testis, pancreas and, to a lesser extent, in brain and kidney (FIG. 3b). The expression of the 4.2-kb transcript was very low in adult brain but more marked in 21-week fetal brain. A second transcript of 6.8 kb was detected in heart and skeletal muscle, whereas a third transcript of 2.75 kb was detected in muscle and liver. These additional transcripts could arise from alternative processing or the use of multiple polyadenylation sites. The sequence shown in FIG. 2 corresponds to 4.2-kb messenger RNA, this being the only species detected in HeLa cells.
The putative ALD gene has thus been identified in the distal part of Xq28 which has deletions in one or several exons in 6 of 85 independent ALD patients. Some of these deletions are small and non-overlapping, thus strengthening the conclusion that this gene is indeed involved in ALD. Although the gene coding for VLCFA-CoA synthetase was considered as a candidate gene for ALD, a recently cloned rat gene for long-chain (C12 -C16) acyl-CoA synthetase failed to detect homologous sequences on the X chromosome. The putative ALD gene shows no homology to this latter sequence, or to the three other enzymes involved in peroxisomal β-oxidation. Surprisingly, a very significant sequence identity was found with human and rat 70K PMP. Two putative domains could be identified by hydropathy analysis : an amino-terminal hydrophobic region, which presumably contains six transmembrane segments, and a hydrophilic region containing ATP-binding motifs with striking identity to the ATP-binding region of the human 70K PMP. This sequence is well conserved in the ATP-binding cassette (ABC) family of transporters, which include the multidrug-resistant gene product, the cystic fibrosis transmembrane conductance regulator, and the products of the PSF1 and PSF2 genes, which encode peptide transporters and map in the class II region of the human MHC Complex (FIG. 4b). The conserved region includes the A and B consensus sequences (A; G-X4-G-K-T-X6-I/V; B: R/K-X3-G-X3-L(hydrophobic)4-D) of a nucleotide-binding fold, plus a 12-amino-acid motif highly conserved in ABC proteins (FIG. 4b).
ALD protein must therefore be a member of this superfamily of ABC transporters, which are also involved in transport of proteins, amino acids, inorganic ions and peptides in prokaryotes and eukaryotes. Although the predicted sequence of ALD protein (SEQ ID NO:2) shows significant identity to 70K PMP (SEQ ID NO:3), no homology was found to 35K PMP or to other PMPs required for peroxisome biogenesis in yeast. Although ALD was initially thought to involve a deficiency in peroxisomal VLCFA-CoA synthesase, the predicted sequence of the putative ALD protein rather suggests a protein involved in transport of VLCFA-CoA synthetase into the peroxisomal membrane or a protein that is functionally associated with the VLCFA-CoA synthetase in the peroxisomal membrane. The translocation of acyl-CoA oxidase, the next enzyme or the peroxisomal β-oxidation pathway, requires ATP hydrolysis, whereas the transport of VLCFA across the peroxisomal membrane does not, and neither is it impaired in peroxisomes from ALD patients.
Expression of ALD protein was observed in every tissue tested, but the relationship between ALD protein expression and the abundance of peroxisomes in tissues may not be straight-forward. Perosisomes are particularly abundant in liver and kidney, having an average diameter of 0.2-1 μM. In other tissues, including the brain and fibroblasts, they are less abundant and smaller (0.05-0.2 μM). This abundance and size difference may reflect a distinct membrane and matrix protein compositions of peroxisomes in different tissues. Although ALD is associated with a defective oxidation of VLCFA, this metabolic defect is mainly expressed in brain and adrenal tissues.
ALD is characterized by a striking variation in clinical phenotype. In family Ma, an identical deletion was found in two sibs, a boy who developed cerebral ALD at 8 years, and his brother who developed only very mild adrenal insufficiency at 13 years. Furthermore, deletions were associated with the adult form (patients O and R) as well as with the severe childhood form (patients B and L). These differences suggest that the phenotypic variability of ALD is probably due to secondary factors (possibly immunological) or to the influence of still unidentified modifier genes.
II) Transfer and expression of the cDNA sequence of the ALD gene
1) retroviral vector
A defective murine Moloney vector (Mo-MLV) is used.
The defective vector still contains its cis-sequences such as the LTR sequence or part the thereof sufficient for the transcription and integration, the psi sequence necessary for the encapsidation and the PB sequence for Vital replication. In this vector the viral genes gag, pol, env are at least partly deleted.
The promoter of the ALD gene or advantageously a stronger promoter such as the PGK-1 promoter (phosphoglycerate kinase) or SV10 promoter replace the deleted vital gene.
The CDNA of the ALD gene is cloned within the defective vector, under the control of the promoter, in a chosen restriction site.
The retroviral vector is then introduced in a cell line for encapsidation, which cell line expresses the gag, pol and env vital genes. A cell line like NIH/3T3 previously modified with the helper virus (Danos et al, PNAS, 85, 6460-6465, 1988) is used.
The recombinant construct is introduced by transfection and the cells produce vital particles.
2) Infection of cells
The cells used for the transfer of the cDNA sequence are cultivated and contacted and incubated with the retroviral vector. The infected cells are then amplified sufficiently to be used for the treatment.
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(B) LOCATION: 387..2624                                                   
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                   
GCGGACGGACGCGCCTGGTGCCCCGGGGAGGGGCGCCACCGGGGGAGGAGGAGGAGGAGA60            
AGGTGGAGAGGAAGAGACGCCCCCTCTGCCCGAGACCTCTCAAGGCCCTGACCTCAGGGG120           
CCAGGGCACTGACAGGACAGGAGAGCCAAGTTCCTCCACTTGGGCTGCCCGAAGAGGCCG180           
CGACCCTGGAGGGCCCTGAGCCCACCGCACCAGGGGCCCCAGCACCACCCCGGGGGCCTA240           
AAGCGACAGTCTCAGGGGCCATCGCAAGGTTTCCAGTTGCCTAGACAACAGGCCCAGGGT300           
CAGAGCAACAATCCTTCCAGCCACCTGCCTCAACTGCTGCCCCAGGCACCAGCCCCAGTC360           
CCTACGCGGCAGCCAGCCCAGGTGACATGCCGGTGCTCTCCAGGCCCCGGCCC413                  
MetProValLeuSerArgProArgPro                                               
15                                                                        
TGGCGGGGGAACACGCTGAAGCGCACGGCCGTGCTCCTGGCCCTCGCG461                       
TrpArgGlyAsnThrLeuLysArgThrAlaValLeuLeuAlaLeuAla                          
10152025                                                                  
GCCTATGGAGCCCACAAAGTCTACCCCTTGGTGCGCCAGTGCCTGGCC509                       
AlaTyrGlyAlaHisLysValTyrProLeuValArgGlnCysLeuAla                          
303540                                                                    
CCGGCCAGGGGTCTTCAGGCGCCCGCCGGGGAGCCCACGCAGGAGGCC557                       
ProAlaArgGlyLeuGlnAlaProAlaGlyGluProThrGlnGluAla                          
455055                                                                    
TCCGGGGTCGCGGCGGCCAAAGCTGGCATGAACCGGGTATTCCTGCAG605                       
SerGlyValAlaAlaAlaLysAlaGlyMetAsnArgValPheLeuGln                          
606570                                                                    
CGGCTCCTGTGGCTCCTGCGGCTGCTGTTCCCCCGGGTCCTGTGCCGG653                       
ArgLeuLeuTrpLeuLeuArgLeuLeuPheProArgValLeuCysArg                          
758085                                                                    
GAGACGGGGCTGCTGGCCCTGCACTCGGCCGCCTTGGTGAGCCGCACC701                       
GluThrGlyLeuLeuAlaLeuHisSerAlaAlaLeuValSerArgThr                          
9095100105                                                                
TTCCTGTCGGTGTATGTGGCCCGCCTGGACGGAAGGCTGGCCCGCTGC749                       
PheLeuSerValTyrValAlaArgLeuAspGlyArgLeuAlaArgCys                          
110115120                                                                 
ATCGCCCGCAAGGACCCGCGGGCTTTTGGCTGGCAGCTGCTGCAGTGG797                       
IleAlaArgLysAspProArgAlaPheGlyTrpGlnLeuLeuGlnTrp                          
125130135                                                                 
CTCCTCATCGCCCTCCCTGCTACCTTCGTCAACAGTGCCATCCGTTAC845                       
LeuLeuIleAlaLeuProAlaThrPheValAsnSerAlaIleArgTyr                          
140145150                                                                 
CTGGAGGGCCAACTGGCCCTGTCGTTCCGCAGCCGTCTGGTGGCCCAC893                       
LeuGluGlyGlnLeuAlaLeuSerPheArgSerArgLeuValAlaHis                          
155160165                                                                 
GCCTACCGCCTCTACTTCTCCCAGCAGACCTACTACCGGGTCAGCAAC941                       
AlaTyrArgLeuTyrPheSerGlnGlnThrTyrTyrArgValSerAsn                          
170175180185                                                              
ATGGACGGGCGGCTTCGCAACCCTGACCAGTCTCTGACGGAGGACGTG989                       
MetAspGlyArgLeuArgAsnProAspGlnSerLeuThrGluAspVal                          
190195200                                                                 
GTGGCCTTTGCGGCCTCTGTGGCCCACCTCTACTCCAACCTGACCAAG1037                      
ValAlaPheAlaAlaSerValAlaHisLeuTyrSerAsnLeuThrLys                          
205210215                                                                 
CCACTCCTGGACGTGGCTGTGACTTCCTACACCCTGCTTCGGGCGGCC1085                      
ProLeuLeuAspValAlaValThrSerTyrThrLeuLeuArgAlaAla                          
220225230                                                                 
CGCTCCCGTGGAGCCGGCACAGCCTGGCCCTCGGCCATCGCCGGCCTC1133                      
ArgSerArgGlyAlaGlyThrAlaTrpProSerAlaIleAlaGlyLeu                          
235240245                                                                 
GTGGTGTTCCTCACGGCCAACGTGCTGCGGGCCTTCTCGCCCAAGTTC1181                      
ValValPheLeuThrAlaAsnValLeuArgAlaPheSerProLysPhe                          
250255260265                                                              
GGGGAGCTGGTGGCAGAGGAGGCGCGGCGGAAGGGGGAGCTGCGCTAC1229                      
GlyGluLeuValAlaGluGluAlaArgArgLysGlyGluLeuArgTyr                          
270275280                                                                 
ATGCACTCGCGTGTGGTGGCCAACTCGGAGGAGATCGCCTTCTATGGG1277                      
MetHisSerArgValValAlaAsnSerGluGluIleAlaPheTyrGly                          
285290295                                                                 
GGCCATGAGGTGGAGCTGGCCCTGCTACAGCGCTCCTACCAGGACCTG1325                      
GlyHisGluValGluLeuAlaLeuLeuGlnArgSerTyrGlnAspLeu                          
300305310                                                                 
GCCTCGCAGATCAACCTCATCCTTCTGGAACGCCTGTGGTATGTTATG1373                      
AlaSerGlnIleAsnLeuIleLeuLeuGluArgLeuTrpTyrValMet                          
315320325                                                                 
CTGGAGCAGTTCCTCATGAAGTATGTGTGGAGCGCCTCGGGCCTGCTC1421                      
LeuGluGlnPheLeuMetLysTyrValTrpSerAlaSerGlyLeuLeu                          
330335340345                                                              
ATGGTGGCTGTCCCCATCATCACTGCCACTGGCTACTCAGAGTCAGAT1469                      
MetValAlaValProIleIleThrAlaThrGlyTyrSerGluSerAsp                          
350355360                                                                 
GCAGAGGCCGTGAAGAAGGCAGCCTTGGAAAAGAAGGAGGAGGAGCTG1517                      
AlaGluAlaValLysLysAlaAlaLeuGluLysLysGluGluGluLeu                          
365370375                                                                 
GTGAGCGAGCGCACAGAAGCCTTCACTATTGCCCGCAACCTCCTGACA1565                      
ValSerGluArgThrGluAlaPheThrIleAlaArgAsnLeuLeuThr                          
380385390                                                                 
GCGGCTGCAGATGCCATTGAGCGGATCATGTCGTCGTACAAGGAGGTG1613                      
AlaAlaAlaAspAlaIleGluArgIleMetSerSerTyrLysGluVal                          
395400405                                                                 
ACGGAGCTGGCTGGCTACACAGCCCGGGTGCACGAGATGTTCCAGGTA1661                      
ThrGluLeuAlaGlyTyrThrAlaArgValHisGluMetPheGlnVal                          
410415420425                                                              
TTTGAAGATGTTCAGCGCTGTCACTTCAAGAGGCCCAGGGAGCTAGAG1709                      
PheGluAspValGlnArgCysHisPheLysArgProArgGluLeuGlu                          
430435440                                                                 
GACGCTCAGGCGGGGTCTGGGACCATAGGCCGGTCTGGTGTCCGTGTG1757                      
AspAlaGlnAlaGlySerGlyThrIleGlyArgSerGlyValArgVal                          
445450455                                                                 
GAGGGCCCCCTGAAGATCCGAGGCCAGGTGGTGGATGTGGAACAGGGG1805                      
GluGlyProLeuLysIleArgGlyGlnValValAspValGluGlnGly                          
460465470                                                                 
ATCATCTGCGAGAACATCCCCATCGTCACGCCCTCAGGAGAGGTGGTG1853                      
IleIleCysGluAsnIleProIleValThrProSerGlyGluValVal                          
475480485                                                                 
GTGGCCAGCCTCAACATCAGGGTGGAGGAAGGCATGCATCTGCTCATC1901                      
ValAlaSerLeuAsnIleArgValGluGluGlyMetHisLeuLeuIle                          
490495500505                                                              
ACAGGCCCCAATGGCTGCGGCAAGAGCTCCCTGTTCCGGATCCTGGGT1949                      
ThrGlyProAsnGlyCysGlyLysSerSerLeuPheArgIleLeuGly                          
510515520                                                                 
GGGCTCTGGCCCACGTACGGTGGTGTGCTCTACAAGCCCCCACCCCAG1997                      
GlyLeuTrpProThrTyrGlyGlyValLeuTyrLysProProProGln                          
525530535                                                                 
CGCATGTTCTACATCCCGCAGAGGCCCTACATGTCTGTGGGCTCCCTG2045                      
ArgMetPheTyrIleProGlnArgProTyrMetSerValGlySerLeu                          
540545550                                                                 
CGTGACCAGGTGATCTACCCGGACTCAGTGGAGGACATGCAAAGGAAG2093                      
ArgAspGlnValIleTyrProAspSerValGluAspMetGlnArgLys                          
555560565                                                                 
GGCTACTCGGAGCAGGACCTGGAAGCCATCCTGGACGTCGTGCACCTG2141                      
GlyTyrSerGluGlnAspLeuGluAlaIleLeuAspValValHisLeu                          
570575580585                                                              
CACCACATCCTGCAGCGGGAGGGAGGTTGGGAGGCTATGTGTGACTGG2189                      
HisHisIleLeuGlnArgGluGlyGlyTrpGluAlaMetCysAspTrp                          
590595600                                                                 
AAGGACGTCCTGTCGGGTGGCGAGAAGCAGAGAATCGGCATGGCCCGC2237                      
LysAspValLeuSerGlyGlyGluLysGlnArgIleGlyMetAlaArg                          
605610615                                                                 
ATGTTCTACCACAGGCCCAAGTACGCCCTCCTGGATGAATGCACCAGC2285                      
MetPheTyrHisArgProLysTyrAlaLeuLeuAspGluCysThrSer                          
620625630                                                                 
GCCGTGAGCATCGACGTGGAAGGCAAGATCTTCCAGGCGGCCAAGGAC2333                      
AlaValSerIleAspValGluGlyLysIlePheGlnAlaAlaLysAsp                          
635640645                                                                 
GCGGGCATTGCCCTGCTCTCCATCACCCACCGGCCCTCCCTGTGGAAA2381                      
AlaGlyIleAlaLeuLeuSerIleThrHisArgProSerLeuTrpLys                          
650655660665                                                              
TACCACACACACTTGCTACAGTTCGATGGGGAGGGCGGCTGGAAGTTC2429                      
TyrHisThrHisLeuLeuGlnPheAspGlyGluGlyGlyTrpLysPhe                          
670675680                                                                 
GAGAAGCTGGACTCAGCTGCCCGCCTGAGCCTGACGGAGGAGAAGCAG2477                      
GluLysLeuAspSerAlaAlaArgLeuSerLeuThrGluGluLysGln                          
685690695                                                                 
CGGCTGGAGCAGCAGCTGGCGGGCATTCCCAAGATGCAGCGGCGCCTC2525                      
ArgLeuGluGlnGlnLeuAlaGlyIleProLysMetGlnArgArgLeu                          
700705710                                                                 
CAGGAGCTCTGCCAGATCCTGGGCGAGGCCGTGGCCCCAGCGCATGTG2573                      
GlnGluLeuCysGlnIleLeuGlyGluAlaValAlaProAlaHisVal                          
715720725                                                                 
CCGGCACCTAGCCCGCAAGGCCCTGGTGGCCTCCAGGGTGCCTCCACC2621                      
ProAlaProSerProGlnGlyProGlyGlyLeuGlnGlyAlaSerThr                          
730735740745                                                              
TGACACAACCGTCCCCGGCCCCTGCCCCGCCCCCAAGCTCGGATCACATGAAGGAGACAG2681          
CAGCACCCACCCATGCACGCACCCCGCCCCTGCATGCCTGGCCCCTCCTCCTAGAAAACC2741          
CTTCCCGCC2750                                                             
(2) INFORMATION FOR SEQ ID NO:2:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 745 amino acids                                               
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                   
MetProValLeuSerArgProArgProTrpArgGlyAsnThrLeuLys                          
151015                                                                    
ArgThrAlaValLeuLeuAlaLeuAlaAlaTyrGlyAlaHisLysVal                          
202530                                                                    
TyrProLeuValArgGlnCysLeuAlaProAlaArgGlyLeuGlnAla                          
354045                                                                    
ProAlaGlyGluProThrGlnGluAlaSerGlyValAlaAlaAlaLys                          
505560                                                                    
AlaGlyMetAsnArgValPheLeuGlnArgLeuLeuTrpLeuLeuArg                          
65707580                                                                  
LeuLeuPheProArgValLeuCysArgGluThrGlyLeuLeuAlaLeu                          
859095                                                                    
HisSerAlaAlaLeuValSerArgThrPheLeuSerValTyrValAla                          
100105110                                                                 
ArgLeuAspGlyArgLeuAlaArgCysIleAlaArgLysAspProArg                          
115120125                                                                 
AlaPheGlyTrpGlnLeuLeuGlnTrpLeuLeuIleAlaLeuProAla                          
130135140                                                                 
ThrPheValAsnSerAlaIleArgTyrLeuGluGlyGlnLeuAlaLeu                          
145150155160                                                              
SerPheArgSerArgLeuValAlaHisAlaTyrArgLeuTyrPheSer                          
165170175                                                                 
GlnGlnThrTyrTyrArgValSerAsnMetAspGlyArgLeuArgAsn                          
180185190                                                                 
ProAspGlnSerLeuThrGluAspValValAlaPheAlaAlaSerVal                          
195200205                                                                 
AlaHisLeuTyrSerAsnLeuThrLysProLeuLeuAspValAlaVal                          
210215220                                                                 
ThrSerTyrThrLeuLeuArgAlaAlaArgSerArgGlyAlaGlyThr                          
225230235240                                                              
AlaTrpProSerAlaIleAlaGlyLeuValValPheLeuThrAlaAsn                          
245250255                                                                 
ValLeuArgAlaPheSerProLysPheGlyGluLeuValAlaGluGlu                          
260265270                                                                 
AlaArgArgLysGlyGluLeuArgTyrMetHisSerArgValValAla                          
275280285                                                                 
AsnSerGluGluIleAlaPheTyrGlyGlyHisGluValGluLeuAla                          
290295300                                                                 
LeuLeuGlnArgSerTyrGlnAspLeuAlaSerGlnIleAsnLeuIle                          
305310315320                                                              
LeuLeuGluArgLeuTrpTyrValMetLeuGluGlnPheLeuMetLys                          
325330335                                                                 
TyrValTrpSerAlaSerGlyLeuLeuMetValAlaValProIleIle                          
340345350                                                                 
ThrAlaThrGlyTyrSerGluSerAspAlaGluAlaValLysLysAla                          
355360365                                                                 
AlaLeuGluLysLysGluGluGluLeuValSerGluArgThrGluAla                          
370375380                                                                 
PheThrIleAlaArgAsnLeuLeuThrAlaAlaAlaAspAlaIleGlu                          
385390395400                                                              
ArgIleMetSerSerTyrLysGluValThrGluLeuAlaGlyTyrThr                          
405410415                                                                 
AlaArgValHisGluMetPheGlnValPheGluAspValGlnArgCys                          
420425430                                                                 
HisPheLysArgProArgGluLeuGluAspAlaGlnAlaGlySerGly                          
435440445                                                                 
ThrIleGlyArgSerGlyValArgValGluGlyProLeuLysIleArg                          
450455460                                                                 
GlyGlnValValAspValGluGlnGlyIleIleCysGluAsnIlePro                          
465470475480                                                              
IleValThrProSerGlyGluValValValAlaSerLeuAsnIleArg                          
485490495                                                                 
ValGluGluGlyMetHisLeuLeuIleThrGlyProAsnGlyCysGly                          
500505510                                                                 
LysSerSerLeuPheArgIleLeuGlyGlyLeuTrpProThrTyrGly                          
515520525                                                                 
GlyValLeuTyrLysProProProGlnArgMetPheTyrIleProGln                          
530535540                                                                 
ArgProTyrMetSerValGlySerLeuArgAspGlnValIleTyrPro                          
545550555560                                                              
AspSerValGluAspMetGlnArgLysGlyTyrSerGluGlnAspLeu                          
565570575                                                                 
GluAlaIleLeuAspValValHisLeuHisHisIleLeuGlnArgGlu                          
580585590                                                                 
GlyGlyTrpGluAlaMetCysAspTrpLysAspValLeuSerGlyGly                          
595600605                                                                 
GluLysGlnArgIleGlyMetAlaArgMetPheTyrHisArgProLys                          
610615620                                                                 
TyrAlaLeuLeuAspGluCysThrSerAlaValSerIleAspValGlu                          
625630635640                                                              
GlyLysIlePheGlnAlaAlaLysAspAlaGlyIleAlaLeuLeuSer                          
645650655                                                                 
IleThrHisArgProSerLeuTrpLysTyrHisThrHisLeuLeuGln                          
660665670                                                                 
PheAspGlyGluGlyGlyTrpLysPheGluLysLeuAspSerAlaAla                          
675680685                                                                 
ArgLeuSerLeuThrGluGluLysGlnArgLeuGluGlnGlnLeuAla                          
690695700                                                                 
GlyIleProLysMetGlnArgArgLeuGlnGluLeuCysGlnIleLeu                          
705710715720                                                              
GlyGluAlaValAlaProAlaHisValProAlaProSerProGlnGly                          
725730735                                                                 
ProGlyGlyLeuGlnGlyAlaSerThr                                               
740745                                                                    
(2) INFORMATION FOR SEQ ID NO:3:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 659 amino acids                                               
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                   
MetAlaAlaPheSerLysTyrLeuThrAlaArgAsnSerSerLeuAla                          
151015                                                                    
GlyAlaAlaPheLeuLeuLeuCysLeuLeuHisLysArgArgArgAla                          
202530                                                                    
LeuGlyLeuHisGlyLysLysSerGlyLysProProLeuGlnAsnAsn                          
354045                                                                    
GluLysGluGlyLysLysGluArgAlaValValAspLysValPhePhe                          
505560                                                                    
SerArgLeuIleGlnIleLeuLysIleMetValProArgThrPheCys                          
65707580                                                                  
LysGluThrGlyTyrLeuValLeuIleAlaValMetLeuValSerArg                          
859095                                                                    
ThrTyrCysAspValTrpMetIleGlnAsnGlyThrLeuIleGluSer                          
100105110                                                                 
GlyIleIleGlyArgSerArgLysAspPheLysArgTyrLeuLeuAsn                          
115120125                                                                 
PheIleAlaAlaMetProLeuIleSerLeuValAsnAsnPheLeuLys                          
130135140                                                                 
TyrGlyLeuAsnGluLeuLysLeuCysPheArgValArgLeuThrLys                          
145150155160                                                              
TyrLeuTyrGluGluTyrLeuGlnAlaPheThrTyrTyrLysLysGly                          
165170175                                                                 
AsnLeuAspAsnArgIleAlaAsnProAspGlnLeuLeuThrGlnAsp                          
180185190                                                                 
ValGluLysPheCysAsnSerValValAspLeuTyrSerAsnLeuSer                          
195200205                                                                 
LysProPheLeuAspIleValLeuTyrIlePheLysLeuThrSerAla                          
210215220                                                                 
IleGlyAlaGlnGlyProAlaSerMetMetAlaTyrLeuValValSer                          
225230235240                                                              
GlyLeuPheLeuThrArgLeuArgArgProIleGlyLysMetThrIle                          
245250255                                                                 
ThrGluGlnLysTyrGluGlyGluTyrArgTyrValAsnSerArgLeu                          
260265270                                                                 
IleThrAsnSerGluGluIleAlaPheTyrAsnGlyAsnLysArgGlu                          
275280285                                                                 
LysGlnThrValHisSerValPheArgLysLeuValGluHisLeuHis                          
290295300                                                                 
AsnPheIleLeuPheArgPheSerMetGlyPheIleAspSerIleIle                          
305310315320                                                              
AlaLysTyrLeuAlaThrValValGlyTyrLeuValValSerArgPro                          
325330335                                                                 
PheLeuAspLeuSerHisProArgHisLeuLysSerThrHisSerGlu                          
340345350                                                                 
LeuLeuGluAspTyrTyrGlnSerGlyArgMetLeuLeuArgMetSer                          
355360365                                                                 
GlnAlaLeuGlyArgIleValLeuAlaGlyArgGluMetThrArgLeu                          
370375380                                                                 
AlaGlyPheThrAlaArgIleThrGluLeuMetGlnValLeuLysAsp                          
385390395400                                                              
LeuAsnHisGlyLysTyrGluArgThrMetValSerGlnGlnGluLys                          
405410415                                                                 
GlyIleGluGlyValGlnValIleProLeuIleProGlyAlaGlyGlu                          
420425430                                                                 
IleIleIleAlaAspAsnIleIleLysPheAspHisValProLeuAla                          
435440445                                                                 
ThrProAsnGlyAspValLeuIleArgAspLeuAsnPheGluValArg                          
450455460                                                                 
SerGlyAlaAsnValLeuIleCysGlyProAsnGlyCysGlyLysSer                          
465470475480                                                              
SerLeuPheArgValLeuGlyGluLeuTrpProLeuPheGlyGlyArg                          
485490495                                                                 
LeuThrLysProGluArgArgLysLeuPheTyrValProGlnArgPro                          
500505510                                                                 
TyrMetThrLeuGlyThrLeuArgAspGlnValIleTyrProAspGly                          
515520525                                                                 
ArgGluAspGlnLysArgLysGlyIleSerAspLeuValGlnLysGlu                          
530535540                                                                 
TyrLeuAspAsnValGlnLeuGlyHisIleLeuGluArgGluGlyGly                          
545550555560                                                              
TrpAspSerValGlnAspTrpMetAspValLeuSerGlyGlyGluLys                          
565570575                                                                 
GlnArgMetAlaMetAlaArgLeuPheTyrHisLysProGlnPheAla                          
580585590                                                                 
IleLeuAspGluCysThrSerAlaValSerValAspValGluGlyTyr                          
595600605                                                                 
IleTyrSerHisCysArgLysValGlyIleThrLeuPheThrValSer                          
610615620                                                                 
HisArgLysSerLeuTrpLysHisHisGluTyrTyrLeuHisMetAsp                          
625630635640                                                              
GlyArgGlyAsnTyrGluPheLysGlnIleThrGluAspThrValGlu                          
645650655                                                                 
PheGlySer                                                                 
(2) INFORMATION FOR SEQ ID NO:4:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 152 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                   
AGGTGGGGCAGGTTGGGGTGCCGGGCACGGAGGGAAGCGTGTGGCAGGGAGGCCCGGGGG60            
CAGGCAGCCGTGAGCGGTGGGGACAGTCTGGGGCGGGCCGGGGCTGATGCCAAAGGTGTG120           
GGCAGGCCATGGGAGAGCCGGGCTGGGGTGGG152                                       
(2) INFORMATION FOR SEQ ID NO:5:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 152 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                   
CACCCAATCGTAACCTCTGGCTCTCGGCCTTCTGATGGCCACCATGGCACAGCGTGTGTG60            
AGTGGCACTGGGAGACCCTGACCATCGCCCCCACGGGAGCTGCCCCTGTGCATGGCCAGG120           
AAGCCTCTCTGTGTCTGTCACCCCCCGCAGGT152                                       
(2) INFORMATION FOR SEQ ID NO:6:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 152 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                   
AGGTGAGACCCAGGGCTCCAAGAGGATCCAGGCCAGGGGCCTGTCCCCCATACCGCTGGG60            
TGCTGAGCTCACGAGGGCCCAACTCAGCCAGCCCGCCGCCCACTTCTGCTGCCGGGGCCA120           
CCGAGGCCCTGCTGCCAGCCTTGATGCTTTCA152                                       
(2) INFORMATION FOR SEQ ID NO:7:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 152 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                   
GCACATAGAGAGAAAGAGAGAGAGAGCTGGTTGCCCCGGCACCATTTGCAGAAGAGCCTC60            
GCCTTTCTCTCCAGCGGCTCATTTTTGACTTTCCGCTGTCTCTGCCCTGCCCCTCCCCGC120           
CCCGCCACCCACCCCTCTGGGGCTTTGCAGAT152                                       
(2) INFORMATION FOR SEQ ID NO:8:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 97 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                   
AGGAGGTACCCCTGGCCCAGCCCCACCCTTGCCATCCTTGCCATGCTTCTCTCCCTGCAA60            
CTGGCAGGGGCTGAGCCAGGGTCACCCTCCCTCAGGT97                                   
(2) INFORMATION FOR SEQ ID NO:9:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 152 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                   
AGGTAAGGCTGTCCCCTCCCTATGAGTGACCCCGCCCCTGCTGCTGCTGCAGGTGCTGAC60            
CTGCTGCCCCAGCTCCTCCTATTCCCGCTCCCTCACTCAGGGACCTCCATGTGCTTCTGG120           
CCCATCCCAGTCCACCCAGGACGGGAGGGCTG152                                       
(2) INFORMATION FOR SEQ ID NO:10:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 152 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                  
CTGGACCACAGGCTGCTGGTCAGGAACCAGCTGGCATGCTGCCAGGGATGGGAATGAGGG60            
CGTGCAGCCAGGGGCACGCAGACTCCCCAGAATGCAGAGGGGTCGCCACCACTCCCTCTC120           
CACCCCAGCCCCGCTGTGCTGTCTCTGCAGGC152                                       
(2) INFORMATION FOR SEQ ID NO:11:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 152 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                  
GGGTAGGTCCAGCGGGGAGGGCGCCAGCCACGCACATATGCAAGCCTCAGCCCTTGGCTT60            
CCCGCCTGTCTGTGCTGGCAACAGCCATTGTCCCTAGATGTACGTGGCAGGTGGGCCAAG120           
GTCAAGGTGAGAGACCAACGTGTCTCTGACTG152                                       
(2) INFORMATION FOR SEQ ID NO:12:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 152 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                  
TCCCCCAGGCCCTGCTGTCCCTTATCAAGAGATCAAGAATGGCCTGCGTGCTGGCCTCGG60            
GCATTGGGAGCCTCTCAAGGCTGGTCAGGAGGCCATAGGGTACGGGAAGGGGCCTGCGCT120           
CTCTGGCGTCAGCGGCTGTTGCCCCTGCAGGT152                                       
(2) INFORMATION FOR SEQ ID NO:13:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 340 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                  
AGGTAAGGAAGCCCGTGCGCCTCTCCTCCACCTCTTCCTGCCTGTGCGCTCACACATGGC60            
TTCCTGCAGAGGCCCAGGAAGTGGTGAAGAGTCAGCACCTCAGGAGAGGACACTGAGGCA120           
CTGTCCCCAGAGCCAGAGACGGGCTGTGGTTCCTGCTCCCTCCAAACCCGCCCGATCCAC180           
TGCCCTGTTTTGGATCTGTGTGGGGTGTGTGCACGGGCGGCGATGTGAGCGTGTGGATGC240           
GTGTGAGCGTGGCATGTGGACACTGCCTGGGAGGCGCAGAGTATCTTGGGGGAGGCAGAG300           
CCGGCCCTTCCCTCCGTGGACACCCAGCTTTCCCACAGGC340                               
(2) INFORMATION FOR SEQ ID NO:14:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 152 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                  
AGGTAGGAGGCCTGGGGCTGGCAGCCACCCTTTGTCCCACCCTGGCCTCTCCCTTGGCCT60            
CCAGGGAGTGAAGATTACCTCAACATCCAGAGTCTAAAGTGCCAGGTGCCACGGGGCGGG120           
GCAGAGGCTGCTACCAGGGAGGACCAACACCA152                                       
(2) INFORMATION FOR SEQ ID NO:15:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 152 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                  
ATGATTAATGCCTGTCAGACAGACAAGGACGCAGAGGCACAGGGGCCCTGTCGTCACAGC60            
TAGCTCATTCCCGCAGCTCCCCCAGCTCCCCGGCTGGCCCCCGGGTCTGGGTGCTGGTGG120           
AACTGAGCCAAGACCATTGCCCCCGCCTAGGT152                                       
(2) INFORMATION FOR SEQ ID NO:16:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 153 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                  
AGGTGAGCACTCCGGACCGGCAGGCTCCCTGGGGTCCCCTGGAAGGGGAAGTAGCAGCTG60            
TGGGGAGGCCTGGGCTCAGTGGAGCCTGAGCCGGGCTGGGGTGTTGGGCCCTGGAGGGTG120           
CACAGACTCTCCTCTCGGCCCGGACCCCCAGGC153                                      
(2) INFORMATION FOR SEQ ID NO:17:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 146 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                  
TGGTAGGTGCCCTGTCTCCCTGCCTGGGGTCGGTGGGAGTGCCTGCCTGAGGGGAGGAGG60            
TGGCCTGGCGGGCCCGGCAGCAGCAGGCGGCTGTCATCAGCAGCCCCCGTGCCGTGCCCC120           
TGACCCTGTCCCTCTCCTGGCCAGGA146                                             
(2) INFORMATION FOR SEQ ID NO:18:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 1441 base pairs                                               
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                  
GCGGAGCGGACGGACGCGCCTGGTGCCCCGGGGAGGGGCGCCACCGGGGGAGGAGGAGGA60            
GGAGAAGGTGGAGAGGAAGAGACGCCCCCTCTGCCCGAGACCTCTCAAGGCCCTGACCTC120           
AGGGGCCAGGGCACTGACAGGACAGGAGAGCCAAGTTCCTCCACTTGGGCTGCCCGAAGA180           
GGCCGCGACCCTGGAGGGCCCTGAGCCCACCGCACCAGGGGCCCCAGCACCACCCCGGGG240           
GCCTAAAGCGACAGTCTCAGGGGCCATCGCAAGGTTTCCAGTTGCCTAGACAACAGGCCC300           
AGGGTCAGAGCAACAATCCTTCCAGCCACCTGCCTCAACTGCTGCCCCAGGCACCAGCCC360           
CAGTCCCTACGCGGCAGCCAGCCCAGGTGACATGCCGGTGCTCTCCAGGCCCCGGCCCTG420           
GCGGGGGAACACGCTGAAGCGCACGGCCGTGCTCCTGGCCCTCGCGGCCTATGGAGCCCA480           
CAAAGTCTACCCCTTGGTGCGCCAGTGCCTGGCCCCGGCCAGGGGTCTTCAGGCGCCCGC540           
CGGGGAGCCCACGCAGGAGGCCTCCGGGGTCGCGGCGGCCAAAGCTGGCATGAACCGGGT600           
ATTCCTGCAGCGGCTCCTGTGGCTCCTGCGGCTGCTGTTCCCCCGGGTCCTGTGCCGGGA660           
GACGGGGCTGCTGGCCCTGCACTCGGCCGCCTTGGTGAGCCGCACCTTCCTGTCGGTGTA720           
TGTGGCCCGCCTGGACGGAAGGCTGGCCCGCTGCATCGCCCGCAAGGACCCGCGGGCTTT780           
TGGCTGGCAGCTGCTGCAGTGGCTCCTCATCGCCCTCCCTGCTACCTTCGTCAACAGTGC840           
CATCCGTTACCTGGAGGGCCAACTGGCCCTGTCGTTCCGCAGCCGTCTGGTGGCCCACGC900           
CTACCGCCTCTACTTCTCCCAGCAGACCTACTACCGGGTCAGCAACATGGACGGGCGGCT960           
TCGCAACCCTGACCAGTCTCTGACGGAGGACGTGGTGGCCTTTGCGGCCTCTGTGGCCCA1020          
CCTCTACTCCAACCTGACCAAGCCACTCCTGGACGTGGCTGTGACTTCCTACACCCTGCT1080          
TCGGGCGGCCCGCTCCCGTGGAGCCGGCACAGCCTGGCCCTCGGCCATCGCCGGCCTCGT1140          
GGTGTTCCTCACGGCCAACGTGCTGCGGGCCTTCTCGCCCAAGTTCGGGGAGCTGGTGGC1200          
AGAGGAGGCGCGGCGGAAGGGGGAGCTGCGCTACATGCACTCGCGTGTGGTGGCCAACTC1260          
GGAGGAGATCGCCTTCTATGGGGGCCATGAGGTGGGGCAGGTTGGGGTGCCGGGCACGGA1320          
GGGAAGCGTGTGGCAGGGAGGCCCGGGGGCAGGCAGCCGTGAGCGGTGGGGACAGTCTGG1380          
GGCGGGCCGGGGCTGATGCCAAAGGTGTGGGCAGGCCATGGGAGAGCCGGGCTGGGGTGG1440          
G1441                                                                     
(2) INFORMATION FOR SEQ ID NO:19:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 481 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                  
CACCCAATCGTAACCTCTGGCTCTCGGCCTTCTGATGGCCACCATGGCACAGCGTGTGTG60            
AGTGGCACTGGGAGACCCTGACCATCGCCCCCACGGGAGCTGCCCCTGTGCATGGCCAGG120           
AAGCCTCTCTGTGTCTGTCACCCCCCGCAGGTGGAGCTGGCCCTGCTACAGCGCTCCTAC180           
CAGGACCTGGCCTCGCAGATCAACCTCATCCTTCTGGAACGCCTGTGGTATGTTATGCTG240           
GAGCAGTTCCTCATGAAGTATGTGTGGAGCGCCTCGGGCCTGCTCATGGTGGCTGTCCCC300           
ATCATCACTGCCACTGGCTACTCAGAGTCAGGTGAGACCCAGGGCTCCAAGAGGATCCAG360           
GCCAGGGGCCTGTCCCCCATACCGCTGGGTGCTGAGCTCACGAGGGCCCAACTCAGCCAG420           
CCCGCCGCCCACTTCTGCTGCCGGGGCCACCGAGGCCCTGCTGCCAGCCTTGATGCTTTC480           
A481                                                                      
(2) INFORMATION FOR SEQ ID NO:20:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 702 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                  
GCACATAGAGAGAAAGAGAGAGAGAGCTGGTTGCCCCGGCACCATTTGCAGAAGAGCCTC60            
GCCTTTCTCTCCAGCGGCTCATTTTTGACTTTCCGCTGTCTCTGCCCTGCCCCTCCCCGC120           
CCCGCCACCCACCCCTCTGGGGCTTTGCAGATGCAGAGGCCGTGAAGAAGGCAGCCTTGG180           
AAAAGAAGGAGGAGGAGCTGGTGAGCGAGCGCACAGAAGCCTTCACTATTGCCCGCAACC240           
TCCTGACAGCGGCTGCAGATGCCATTGAGCGGATCATGTCGTCGTACAAGGAGGTACCCC300           
TGGCCCAGCCCCACCCTTGCCATCCTTGCCATGCTTCTCTCCCTGCAACTGGCAGGGGCT360           
GAGCCAGGGTCACCCTCCCTCAGGTGACGGAGCTGGCTGGCTACACAGCCCGGGTGCACG420           
AGATGTTCCAGGTATTTGAAGATGTTCAGCGCTGTCACTTCAAGAGGCCCAGGGAGCTAG480           
AGGACGCTCAGGCGGGGTCTGGGACCATAGGCCGGTCTGGTGTCCGTGTGGAGGGCCCCC540           
TGAAGATCCGAGGTAAGGCTGTCCCCTCCCTATGAGTGACCCCGCCCCTGCTGCTGCTGC600           
AGGTGCTGACCTGCTGCCCCAGCTCCTCCTATTCCCGCTCCCTCACTCAGGGACCTCCAT660           
GTGCTTCTGGCCCATCCCAGTCCACCCAGGACGGGAGGGCTG702                             
(2) INFORMATION FOR SEQ ID NO:21:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 395 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                  
CTGGACCACAGGCTGCTGGTCAGGAACCAGCTGGCATGCTGCCAGGGATGGGAATGAGGG60            
CGTGCAGCCAGGGGCACGCAGACTCCCCAGAATGCAGAGGGGTCGCCACCACTCCCTCTC120           
CACCCCAGCCCCGCTGTGCTGTCTCTGCAGGCCAGGTGGTGGATGTGGAACAGGGGATCA180           
TCTGCGAGAACATCCCCATCGTCACGCCCTCAGGAGAGGTGGTGGTGGCCAGCCTCAACA240           
TCAGGGTAGGTCCAGCGGGGAGGGCGCCAGCCACGCACATATGCAAGCCTCAGCCCTTGG300           
CTTCCCGCCTGTCTGTGCTGGCAACAGCCATTGTCCCTAGATGTACGTGGCAGGTGGGCC360           
AAGGTCAAGGTGAGAGACCAACGTGTCTCTGACTG395                                    
(2) INFORMATION FOR SEQ ID NO:22:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 928 base pairs                                                
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                  
TCCCCCAGGCCCTGCTGTCCCTTATCAAGAGATCAAGAATGGCCTGCGTGCTGGCCTCGG60            
GCATTGGGAGCCTCTCAAGGCTGGTCAGGAGGCCATAGGGTACGGGAAGGGGCCTGCGCT120           
CTCTGGCGTCAGCGGCTGTTGCCCCTGCAGGTGGAGGAAGGCATGCATCTGCTCATCACA180           
GGCCCCAATGGCTGCGGCAAGAGCTCCCTGTTCCGGATCCTGGGTGGGCTCTGGCCCACG240           
TACGGTGGTGTGCTCTACAAGCCCCCACCCCAGCGCATGTTCTACATCCCGCAGAGGTAA300           
GGAAGCCCGTGCGCCTCTCCTCCACCTCTTCCTGCCTGTGCGCTCACACATGGCTTCCTG360           
CAGAGGCCCAGGAAGTGGTGAAGAGTCAGCACCTCAGGAGAGGACACTGAGGCACTGTCC420           
CCAGAGCCAGAGACGGGCTGTGGTTCCTGCTCCCTCCAAACCCGCCCGATCCACTGCCCT480           
GTTTTGGATCTGTGTGGGGTGTGTGCACGGGCGGCGATGTGAGCGTGTGGATGCGTGTGA540           
GCGTGGCATGTGGACACTGCCTGGGAGGCGCAGAGTATCTTGGGGGAGGCAGAGCCGGCC600           
CTTCCCTCCGTGGACACCCAGCTTTCCCACAGGCCCTACATGTCTGTGGGCTCCCTGCGT660           
GACCAGGTGATCTACCCGGACTCAGTGGAGGACATGCAAAGGAAGGGCTACTCGGAGCAG720           
GACCTGGAAGCCATCCTGGACGTCGTGCACCTGCACCACATCCTGCAGCGGGAGGGAGGT780           
AGGAGGCCTGGGGCTGGCAGCCACCCTTTGTCCCACCCTGGCCTCTCCCTTGGCCTCCAG840           
GGAGTGAAGATTACCTCAACATCCAGAGTCTAAAGTGCCAGGTGCCACGGGGCGGGGCAG900           
AGGCTGCTACCAGGGAGGACCAACACCA928                                           
(2) INFORMATION FOR SEQ ID NO:23:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 1025 base pairs                                               
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                  
ATGATTAATGCCTGTCAGACAGACAAGGACGCAGAGGCACAGGGGCCCTGTCGTCACAGC60            
TAGCTCATTCCCGCAGCTCCCCCAGCTCCCCGGCTGGCCCCCGGGTCTGGGTGCTGGTGG120           
AACTGAGCCAAGACCATTGCCCCCGCCTAGGTTGGGAGGCTATGTGTGACTGGAAGGACG180           
TCCTGTCGGGTGGCGAGAAGCAGAGAATCGGCATGGCCCGCATGTTCTACCACAGGTGAG240           
CACTCCGGACCGGCAGGCTCCCTGGGGTCCCCTGGAAGGGGAAGTAGCAGCTGTGGGGAG300           
GCCTGGGCTCAGTGGAGCCTGAGCCGGGCTGGGGTGTTGGGCCCTGGAGGGTGCACAGAC360           
TCTCCTCTCGGCCCGGACCCCCAGGCCCAAGTACGCCCTCCTGGATGAATGCACCAGCGC420           
CGTGAGCATCGACGTGGAAGGCAAGATCTTCCAGGCGGCCAAGGACGCGGGCATTGCCCT480           
GCTCTCCATCACCCACCGGCCCTCCCTGTGGTAGGTGCCCTGTCTCCCTGCCTGGGGTCG540           
GTGGGAGTGCCTGCCTGAGGGGAGGAGGTGGCCTGGCGGGCCCGGCAGCAGCAGGCGGCT600           
GTCATCAGCAGCCCCCGTGCCGTGCCCCTGACCCTGTCCCTCTCCTGGCCAGGAAATACC660           
ACACACACTTGCTACAGTTCGATGGGGAGGGCGGCTGGAAGTTCGAGAAGCTGGACTCAG720           
CTGCCCGCCTGAGCCTGACGGAGGAGAAGCAGCGGCTGGAGCAGCAGCTGGCGGGCATTC780           
CCAAGATGCAGCGGCGCCTCCAGGAGCTCTGCCAGATCCTGGGCGAGGCCGTGGCCCCAG840           
CGCATGTGCCGGCACCTAGCCCGCAAGGCCCTGGTGGCCTCCAGGGTGCCTCCACCTGAC900           
ACAACCGTCCCCGGCCCCTGCCCCGCCCCCAAGCTCGGATCACATGAAGGAGACAGCAGC960           
ACCCACCCATGCACGCACCCCGCCCCTGCATGCCTGGCCCCTCCTCCTAGAAAACCCTTC1020          
CCGCC1025                                                                 
__________________________________________________________________________

Claims (5)

We claim:
1. An isolated nucleotide sequence selected from the group consisting of a cDNA having the sequence represented in FIG. 2 (SEQ ID NO:1), an isolated genomic DNA sequence comprising said cDNA, and RNA encoding the protein of SEQ ID NO:2, said sequence being in unmutated form and which, in mutated forms, is responsible for adrenoleukodystrophy or adrenomyelopathy.
2. An isolated nucleotide sequence according to claim 1, having the sequence represented in FIG. 6 (SEQ ID NOS: 4-17) or FIG. 7 (SEQ ID NOS: 8-23).
3. An isolated DNA sequence consisting of a DNA sequence encoding the human adrenoleukodystrophy protein.
4. An isolated DNA sequence according to claim 3, consisting of the sequence represented in FIG. 2 (SEQ ID NO:1).
5. An isolated genomic DNA sequence according to claim 1, which has the restriction sites shown in FIG. 5.
US08/136,277 1993-10-15 1993-10-15 X-linked adrenoleukodystrophy gene and corresponding protein Expired - Lifetime US5644045A (en)

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US08/479,403 US5869039A (en) 1993-10-15 1995-06-07 X-linked adrenoleukodystrophy gene and corresponding protein
US08/835,734 US6013769A (en) 1993-10-15 1997-04-10 X-linked adrenoleukodystrophy gene and corresponding protein

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
US6013769A (en) * 1993-10-15 2000-01-11 Institut National De La Sante Et De La Recherche Medicale X-linked adrenoleukodystrophy gene and corresponding protein
US6949624B1 (en) * 1999-08-03 2005-09-27 The United States Of America As Represented By The Department Of Health And Human Services Cloning of the human nuclear receptor co-repressor gene

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FR2729399B1 (en) * 1995-01-13 1997-04-04 Inst Nat Sante Rech Med RECOMBINANT RETROVIRAL VECTOR FOR CORRECTION OF GENE TRANSFER ADRENOLEUCODYSTROPHY
FR2775694B1 (en) * 1998-03-06 2000-06-09 Gradient Ass TOXICITY TEST

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Title
By H. Moser et al., "Adrenoleukodystrophy: Phenotypic Variability and Implications for Therapy", 1992, pp. 645-664.
By H. Moser et al., Adrenoleukodystrophy: Phenotypic Variability and Implications for Therapy , 1992, pp. 645 664. *
By J. Mosser et al., "Putative X-linked adrenoleukodystrophy gene shares unexprected homology with ABE transporters", Feb. 25, 1993, pp. 726-730, vol. 361, Nature.
By J. Mosser et al., Putative X linked adrenoleukodystrophy gene shares unexprected homology with ABE transporters , Feb. 25, 1993, pp. 726 730, vol. 361, Nature. *
By P. Aubourg et al., "Frequent Alterations of Visual Pigment Genes in Adrenoleukodystrophy", 1988, pp. 408-413, vol. 42, American Journal of Genetics.
By P. Aubourg et al., Frequent Alterations of Visual Pigment Genes in Adrenoleukodystrophy , 1988, pp. 408 413, vol. 42, American Journal of Genetics. *
By R. Feil et al., "Adrenoleukodystrophy: a Complex Chromosomal Rearrangement in the Xq28 Red/Green-Color-Pigment Gene Region Indicates Two Possible Gene Localizations", 1991, vol. 49, pp. 1361-1371,American Journal of Human Genetics.
By R. Feil et al., Adrenoleukodystrophy: a Complex Chromosomal Rearrangement in the Xq28 Red/Green Color Pigment Gene Region Indicates Two Possible Gene Localizations , 1991, vol. 49, pp. 1361 1371,American Journal of Human Genetics. *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6013769A (en) * 1993-10-15 2000-01-11 Institut National De La Sante Et De La Recherche Medicale X-linked adrenoleukodystrophy gene and corresponding protein
US6949624B1 (en) * 1999-08-03 2005-09-27 The United States Of America As Represented By The Department Of Health And Human Services Cloning of the human nuclear receptor co-repressor gene

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